DESCRIPTION (adapted from the applicant's description): The overall goal of this proposal is to elucidate the multiple regulatory pathways required for ligand-mediated control of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel or ryanodine receptor (RYR1). Experiments are designed to test two hypotheses. (1) There are discrete domains on RYR1 that mediate its regulation by the dihydropyridine receptor (DHPR), calcium and other endogenous effector molecules. (2) During strenuous muscle exercise and in fatigue, changes in the intracellular ionic milieu and redox state of RYR1 and possibly phosphorylation elicit changes in channel function. The proposal has four specific aims. Specific aim 1 investigates the regulation of RYR1 by the DHPR and DHPR-derived peptides. Specific aim 2 determines the kinetics of RYR1 regulation and SR lumenal calcium. Specific aim 3 determines the levels of endogenous RYR1 S-nitrosylation and characterizes the regulation of RYR1 by NO and NO-generating compounds. Specific aim 4 defines the relationships between RYR1 protein structure and regulatory mechanisms using site-directed mutagenesis and chimeric constructs. These studies will be conducted in isolated membranes. In addition, purified native and mutant channels will be incorporated into planar lipid bilayers and studied using a ligand binding assay. Experiments will be performed under normal conditions and under conditions mimicking strenuous exercise and fatigue.